Bottom Line:
Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant.Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT

Background: Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway.

Results: Ccr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.

Conclusions: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

Fig2: Mmi1 recruits Ccr4-Not to its mRNA targets. a Relative enrichment of Mmi1 targets in Ccr4 RIP-chip experiments (normalized to actin) in wild-type cells (left) and mmi1 mutants. Each dot corresponds to an individual mRNA, and the not1 mRNA is designated with a star. Horizontal lines the median enrichment of all Mmi1 targets. b Cell extracts were prepared from the indicated strains and mock-treated (−) or treated with RNase (+). Immunoprecipitations were carried out using antibodies against TAP. Samples were probed with antibodies against myc (top) or TAP (bottom). The interaction between TAP-Mmi1 and Red1-myc was used as a positive control. c As in B, but samples were probed with antibodies against a FLAG epitope (top) or TAP (bottom). Similar results were obtained with different epitope tag combinations of Mmi1 and Ccr4.

Mentions:
Mmi1 is thought to bind directly to a well-defined RNA sequence motif [42], while the Ccr4-Not complex is typically recruited to particular mRNAs through sequence-specific RNA-binding proteins [1, 43]. This result raised the possibility that Ccr4-Not is recruited to its RNA targets by Mmi1. To investigate this possibility further, we performed Ccr4 RIP-chip experiments in cells lacking mmi1 (as above, the experiment was performed in a mei4Δ background). As predicted, the interaction between Ccr4 and its RNA targets was completely lost in the absence of Mmi1 (Fig. 2a). By contrast, the mRNA encoding Not1, another component of the Ccr4-Not complex, was enriched in both wild-type and mmi1 mutant backgrounds (Fig. 2a, indicated with a star). The interaction between Ccr4 and the not1 mRNA is likely to represent a cotranslational interaction between Ccr4 and the Not1 nascent peptide [44, 45], and serves as a control for the immunoprecipitation reaction. In addition, the Mmi1 and Ccr4 proteins coprecipitated in an RNA-independent manner, indicating that they are part of the same multiprotein complex (Fig. 2b, c). Taken together, these data indicate that Mmi1 recruits the Ccr4-Not complex to its target mRNAs.Fig. 2

Fig2: Mmi1 recruits Ccr4-Not to its mRNA targets. a Relative enrichment of Mmi1 targets in Ccr4 RIP-chip experiments (normalized to actin) in wild-type cells (left) and mmi1 mutants. Each dot corresponds to an individual mRNA, and the not1 mRNA is designated with a star. Horizontal lines the median enrichment of all Mmi1 targets. b Cell extracts were prepared from the indicated strains and mock-treated (−) or treated with RNase (+). Immunoprecipitations were carried out using antibodies against TAP. Samples were probed with antibodies against myc (top) or TAP (bottom). The interaction between TAP-Mmi1 and Red1-myc was used as a positive control. c As in B, but samples were probed with antibodies against a FLAG epitope (top) or TAP (bottom). Similar results were obtained with different epitope tag combinations of Mmi1 and Ccr4.

Mentions:
Mmi1 is thought to bind directly to a well-defined RNA sequence motif [42], while the Ccr4-Not complex is typically recruited to particular mRNAs through sequence-specific RNA-binding proteins [1, 43]. This result raised the possibility that Ccr4-Not is recruited to its RNA targets by Mmi1. To investigate this possibility further, we performed Ccr4 RIP-chip experiments in cells lacking mmi1 (as above, the experiment was performed in a mei4Δ background). As predicted, the interaction between Ccr4 and its RNA targets was completely lost in the absence of Mmi1 (Fig. 2a). By contrast, the mRNA encoding Not1, another component of the Ccr4-Not complex, was enriched in both wild-type and mmi1 mutant backgrounds (Fig. 2a, indicated with a star). The interaction between Ccr4 and the not1 mRNA is likely to represent a cotranslational interaction between Ccr4 and the Not1 nascent peptide [44, 45], and serves as a control for the immunoprecipitation reaction. In addition, the Mmi1 and Ccr4 proteins coprecipitated in an RNA-independent manner, indicating that they are part of the same multiprotein complex (Fig. 2b, c). Taken together, these data indicate that Mmi1 recruits the Ccr4-Not complex to its target mRNAs.Fig. 2

Bottom Line:
Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant.Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

Affiliation:
Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT

Background: Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway.

Results: Ccr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.

Conclusions: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.